Mesenchymal stem cells-hydrogel-biodegradable or mesenchymal stem cells-hydrogel-nondegradable support composition for alleviating or improving epidermolysis bullosa

ABSTRACT

Provided are a composition and a sheet, including a mesenchymal stem cells-hydrogel-biodegradable support or a mesenchymal stem cells-hydrogel-nondegradable support and a preparing method thereof. More specifically, in the sheet including a mesenchymal stem cells-hydrogel-biodegradable support or a mesenchymal stem cells-hydrogel-nondegradable support according to the present invention, the high-active mesenchymal stem cells may be applied to a wounded part of a patient with epidermolysis bullosa as it is without isolation using proteases, and in the culturing, an extracellular matrix such as collagen, laminin, fibronectin, and elastin secreted from the mesenchymal stem cells is wholly present on the hydrogel to have an advantageous effect that skin reproduction and re-epithelization abilities are significantly excellent as compared with conventional dressing agents used for epidermolysis bullosa.

REFERENCE TO RELATED APPLICATIONS

This is a Continuation-In-Part (CIP) application of PCT/KR2017/003982filed Apr. 12, 2017 which claims priority to KR Application10-2016-0044514 filed Apr. 12, 2016, the disclosure of which beingentirely incorporated herein as reference.

TECHNICAL FIELD

The present invention relates to a composition and a sheet foralleviating or improving epidermolysis bullosa, containing high-activeliving mesenchymal stem cells which are attached and cultured to abiodegradable or nondegradable support by suspending the culturedmesenchymal stem cells in a hydrogel, and a preparing method thereof.Specifically, the present invention is to provide a composition, asheet, and a preparing method thereof capable of expressing a clinicallysignificant effect when applied to a wounded part of epidermolysisbullosa by increasing expression of collagen type VII and laminin-5.Therefore, the composition or sheet may be usefully used for alleviatingor improving symptoms of a patient with epidermolysis bullosa.

BACKGROUND ART

Epidermolysis bullosa is a rare hereditary disease that causes pain inthe skin and mucous membrane due to the blistering easily caused even bylight trauma since the mutation of genes makes proteins which constituteepidermis, epidermis-dermis boundary, and upper papillary dermis.Patients with epidermolysis bullosa have no binding proteins to hold twolayers to form blisters when a small friction occurs on the layer andthen the patient experience pains corresponding to the third-degreeburns. The epidermolysis bullosa includes epidermolysis bullosa simplex(EBS), junctional epidermolysis bullosa (JEB), dystrophic epidermolysisbullosa (DEB), and the like, and most of the patients are known as EBS.Mutation of kelatin generated in the skin is known to be a main cause ofdisease, and it is known that EBS includes autosomal dominantinheritance, JEB includes autosomal recessive inheritance, DEB includesdominant and recessive types.

Up to now, a method of modifying keratin mixtures produced in the skin,methods such as bone marrow transplantation, and the like have beenstudied, but there is no method for the full recovery of the diseaseavailable yet. Main treatment is only made with a symptomatic therapyfor relieving symptoms or complications. It is the most important toprevent blisters from being infected. Most blisters are caused by somepressure or a friction, so, touching should be avoided, and when theblisters are caused, it is known that preventing excessive fluid lossand infection is important while relieving pains and minimizingdiscomfort. Painkillers may also be used to relieve pain while dressingsare performed.

For patients with epidermolysis bullosa, an effective dressing isrecommended to prevent blisters from infections. For example, in case ofEB simplex, a soft silicone type dressing agent, a lipido-colloid typedressing agent, a foam type dressing agent, a hydrogel type dressingagent, a sheet hydrogel type dressing agent, a biosynthetic cellulosetype dressing agent, a bordered type dressing agent, or a powder typedressing agent ise recommended. Also, in case of EBS Dowling Meara, alipido-colloid type dressing agent, a polymer membrane type dressingagent, and a hydrofiber type dressing agent are recommended. Also, incase of junctional EB, a hydrogel impregnated gauze type dressing agent,a hydrofiber type dressing agent, a lipido-colloid type dressing agent,a soft silicone type dressing agent, a polymer membrane type dressingagent, a soft silicon foam type dressing agent, and a soft silicon foamtype dressing agent having a super-adsorbent agent are recommended.Also, in the case of dystrophic EB, a soft silicon type dressing agent,a lipido-colloid type dressing agent, a foam type dressing agent withsoft silicon, a soft silicon foam type dressing agent with asuper-adsorbent agent, a foam type dressing agent, a polymer membranetype dressing agent, a super-adsorption type dressing agent, and aboundary type dressing agent are recommended (see Denyer J, Pillay E.Best practice guidelines for skin and wound care in epidermolysisbullosa. International Consensus. DEBRA, 2012.).

Regarding the dressing for healing wounds, there are patents availablesuch as a patent (Korean Patent Registration No. 345,034) for a dressingagent for healing wounds in a form of a polyurethane foam which enhancesbiocompatibility and promotes absorption of secretions at the woundedsites by introducing a hydrophilic group, a patent (Korean PatentRegistration No. 1,022,884) for a polyurethane foam dressing agentincluding a drug layer, and the like.

In case of dressing agents in the related art are used, it is moreadvantageous in alleviating irritation or facilitating the absorption ofthe eluate from the blister site when applied to a wounded site ofepidermolysis bullosa. However, there are no known techniques for adressing agent exhibits skin regeneration effects without irritationwhen the dressing agent is applied to a wounded site of epidermolysisbullosa patients.

Meanwhile, it is known that collagen type VII or collagen alpha-1 chainis a protein encoded by a COL7A1 gene and associated with dystrophicepidermolysis bullosa when the gene is mutated, and even though there isno mutation, it has been reported that an autoimmune response tocollagen type VII may cause a disease called epidermolysis bullosa.Also, the collagen type VII is also known to interact with laminin-5 andfibronectin. In addition, a study was reported in Italy, in which stemcells containing a calibrated laminin-5 gene were transplanted into afemoral lesion of a patient with junctional epidermolysis bullosathrough skin transplantation to achieve a blister-free clinical effect.Therefore, it can be seen that collagen type VII and laminin-5 play avery important role to alleviate or improve epidermolysis bullosa.

An object of the present invention is to provide a composition and asheet for alleviating or improving epidermolysis bullosa, containinghigh-active living mesenchymal stem cells which are attached andcultured to a biodegradable or nondegradable support by suspending thecultured mesenchymal stem cells in a hydrogel, and a preparing methodthereof. Specifically, an object of the present invention is to providea composition, a sheet, and a preparing method thereof capable ofexpressing a clinically significant effect when applied to a woundedpart of epidermolysis bullosa by increasing expression of collagen typeVII and laminin-5.

DISCLOSURE Technical Problem

The present invention has been made in an effort to provide acomposition or a sheet for alleviating or improving epidermolysisbullosa including high-active mesenchymal stem cells for obtaining aclinically effective therapeutic effect and a preparing method thereof,which is used as a dressing agent for applying mesenchymal stem cellsisolated from any one selected from the group consisting of humanadipose, bone marrow, skin, blood vessels, muscles, brain, blood,placenta, dental pulp and umbilical cord blood to an affected area ofepidermolysis bullosa.

Technical Solution

In order to achieve the objects, the present invention provides acomposition and a sheet for alleviating or improving epidermolysisbullosa, including a mesenchymal stem cells-hydrogel-biodegradable ornondegradable support, more specifically, a mesenchymal stemcells-hydrogel-nondegradable support, and a preparing method thereof.

The mesenchymal stem cells are antilogous or allergenic cells that arepositive for CD29, CD44, CD73, CD90, and CD105 and negative for CD34 andCD45.

In one embodiment according to the present invention, the support mayuse a biodegradable polymer support selected from the group consistingof poly-gamma-glutei acid (PGA), poly lactic acid (PLA), PGA/PLA, viralmesh, human placental membrane, bovine placental membrane, pig collagen,chitin, chatoyant, fibronectin and dextrin, or may use a nondegradablesupport such as sterilized no woven fabrics, polyethylene terephthalate(PET) films, polyethylene (PE) films, polypropylene (PP) films,polyurethane films, net type polyurethane films, or polyurethane coatedwith soft silicon on a single surface, or a combination thereof, forexample, PGA/nonwoven fabric, PLA/nonwoven fabric, and PGA/PLA/nonwovenfabric.

In one embodiment according to the present invention, the hydrogel maybe selected from the group consisting of fibrin glue, hyaluronic acid orderivatives thereof, gelatin, collagen, alginic acid, cellulose andpectin, and in this case, the concentration of fibrinogen forming fibringlue may be 0.5 to 60 mg/mL, specifically 0.5 to 45 mg/mL, morespecifically 0.5 to 30 mg/mL, much more specifically 0.5 to 20 mg/mL,and much more specifically 0.5 to 10 mg/mL.

In one embodiment according to the present invention, in order toprepare the sheet for alleviating or improving epidermolysis bullosa,the method includes proliferating stem cells of 20,000 or more and morespecifically 20,000 to 400,000 per 1 cm² of the support by mixing thestem cells with the hydrogel to evenly coat 1,000 to 10,000 stem cellsper 1 cm² of the support and culturing the stem cells in a mediumcontaining at least one selected from the group consisting of FBS, andbFGF or EGF.

Advantageous Effects

According to the present invention, the composition or sheet foralleviating or improving epidermolysis bullosa, containing themesenchymal stem cells-hydrogel-biodegradable support or nondegradablesupport increases the expression of specifically, collagen type VII andlaminin-5 to express a clinically significant effect when being appliedto the wounded part of epidermolysis bullosa.

Further, in the composition or sheet containing high-active mesenchymalstem cells, the high-active mesenchymal stem cells may be applied to awounded part of a patient with epidermolysis bullosa as it is withoutisolation (selection) using proteases, and in the culturing, anextracellular matrix such as collagen, laminin, fibronectin, and elastinsecreted from the mesenchymal stem cells is wholly present on thehydrogel to have an advantageous effect that skin reproduction andre-epithelization abilities are significantly excellent when thecomposition or sheet is applied to the wounded part of the patient withepidermolysis bullosa as compared with conventional therapeutic agents.

More specifically, the mesenchymal stem cells-hydrogel-support accordingto the present invention keep a form of fibroblasts even in a serum-freemedium, and the cells survive 90% or more after 1 week elapses and thusthe survival time is significantly increased as compared with aconventional stem cell therapeutic agent. Also, when thawing afterfreezing, the shape and the strength of the sheet are kept as it is andthe cells in the sheet also survive 95% or more and long-term freezingat −80° C. is enabled without damage of the cells as a main activeingredient, and even when 1 year or more elapses when long-termfreezing, the cells survive 95% or more when thawing. Further, variousgrowth factors and cytokines promoting cell proliferation andangiogenesis are continuously secreted, various types of extracellularmatrixes containing collagen type VII and laminin-5 are secreted inlarge amount, and the secreted extracellular matrixes are left in thehydrogel and applied to the wounded part to provide various substrates,thereby facilitating treatment of epidermolysis bullosa. Also, even ifthe inflammation occurs in the wounded part without causing the immuneresponse, a secretion amount of TNF-α which is secreted by immune cellsin large amount to increase immunoreactivity is significantly reduced toalleviate the inflammation, thereby helping in healing blisters andinflammation in patients with epidermolysis bullosa. Specifically, theexpression of collagen type VII is significantly increased, therebypromoting tissue reproduction and re-epithelization.

DESCRIPTION OF DRAWINGS

FIG. 1A is a fluorescence microscope photograph obtained by observing aform of mesenchymal stem cells derived from the human adipose which arecultured by mixing a fibrinogen undiluted solution and a fibrin gelprepared by a solution diluted stepwise and stained with AO/EtBr (400×magnification). Concentrations when the final fibrin glue is formed bymixing the dilution rate of the fibrinogen and a cell suspensioncontaining thrombin at a ratio of 1:1 are expressed in parentheses.

FIG. 1B is a graph of absorbance measured by adding WST-1 to mesenchymalstem cells derived from the human adipose which are cultured by mixing afibrinogen undiluted solution and a fibrin gel prepared by a solutiondiluted stepwise.

FIG. 2A is a photograph obtained by culturing a human adipose-derivedmesenchymal stem cells-hydrogel-biodegradable support or nondegradablesupport sheet for 5 days, staining cells in the sheet with AO/EtBr, andobserving the stained cells by a fluorescence microscope, as aphotograph of cells in the sheet.

FIG. 2B is a photograph obtained by freezing at −80° C. and thawing thehuman adipose-derived mesenchymal stem cells-hydrogel-biodegradablesupport or nondegradable support sheet, staining cells in the sheet withAO/EtBr, and observing the stained cells by a fluorescence microscope.

FIG. 3 is a photograph showing survival rate of cells in the sheet afterfreezing the sheet at −80° C. and thawing the sheet for 1, 3, 6, 9, and12 months in order to evaluate long-term storage stability of the humanadipose-derived mesenchymal stem cells-hydrogel-biodegradable support ornondegradable support sheet.

FIG. 4A is a graph showing amounts of VEGF and HGF secreted from thehuman adipose-derived mesenchymal stem cells-hydrogel-biodegradablesupport or nondegradable support sheet which are measured by ELISA.

FIG. 4B is a diagram showing a result of analyzing angiogenesispromoting factors secreted from the human adipose-derived mesenchymalstem cells-hydrogel-biodegradable support or nondegradable support sheetusing a cytokine array kit.

FIG. 5 is a graph showing an amount of TNF-α secreted from peripheralblood mononuclear cells after co-culturing the human adipose-derivedmesenchymal stem cells-hydrogel-biodegradable support or nondegradablesupport sheet with homologous activated peripheral blood mononuclearcells, which is measured by ELISA and converted to a secretioninhibition rate (%) of TNF-α.

FIG. 6A is a diagram showing the results of verifying expression levelsof collagen type VII and laminin among extracellular matrix proteinsexpressed by human adipose-derived mesenchymal stem cells andfibroblasts through Western blot.

FIG. 6B is a graph showing results of verifying the expression level ofcollagen type VII of extracellular matrix proteins expressed in thehuman adipose-derived mesenchymal stem cells by ELISA.

FIG. 6C is a photograph obtained by observing the expression levels ofcollagen type VII and of collagen type I extracellular matrix proteinsexpressed in the human adipose-derived mesenchymal stem cells by afluorescence microscope after immunofluorescence staining.

FIG. 7A is a graph showing amounts of collagen type VII and collagentype I which are extracellular matrix proteins secreted from the sheetaccording to the present invention.

FIG. 7B is a photograph showing the expression levels of collagen typeVII, collagen type I, collagen type V, laminin 5, fibronectin, laminin,and the like which are extracellular matrix proteins included in thesheet according to the present invention by a fluorescence microscopyafter immunofluorescence staining.

FIG. 8 is a graph obtained by comparing an amount of collagen type VIIwhich is an extracellular matrix protein secreted from humanadipose-derived mesenchymal stem cells which are monolayer-cultured withthe sheet according to the present invention.

FIG. 9 is a diagram showing results that expression of collagen type VIIis rapidly increased when human adipose-derived mesenchymal stem cellsare exposed to inflammatory cytokines TGF-β2 and TNF-α secreted from awounded part.

FIG. 10 is a diagram showing results that the sheet according to thepresent invention restores the morphology and adherence of collagen typeVII deficient fibroblasts.

FIG. 11 is a diagram showing results that the sheet according to thepresent invention can regenerate a skin by restoring the mobility of thecollagen type VII deficient fibroblasts.

FIG. 12 is a graph showing changes in the size of the wounded part for atotal of 4 weeks before and after attaching the sheet according to thepresent invention to a patient with epidermolysis bullosa.

DETAILED DESCRIPTION OF THE INVENTION

In order to achieve the objects, the present invention provides acomposition and a sheet for alleviating or improving epidermolysisbullosa, including a mesenchymal stem cells-hydrogel-biodegradable ornondegradable support, and a preparing method thereof.

Hereinafter, the present invention will be described in more detail.

The present invention provides a preparing method of a sheet foralleviating or improving epidermolysis bullosa, including: (a) obtaininga mesenchymal stem cells-hydrogel-support by attaching mesenchymal stemcells to (i) at least one kind of support selected from the groupconsisting of biodegradable supports and nondegradable supports; (ii) atleast two kinds of nondegradable supports; or (iii) a combination of atleast one kind of support and at least one kind of nondegradable supportusing a hydrogel; and (b) culturing the mesenchymal stemcells-hydrogel-support obtained in step (a) in a growth medium. Herein,the growth medium is a medium including fetal bovine serum (FBS) and atleast one factor selected from the group consisting of a basicfibroblast growth factor (bFGF), an epidermal growth factor (EGF), atransforming growth factor beta-1 (TGF-beta1), a platelet-derived growthfactor (PDGF), a vascular endothelial growth factor (VEGF), a hepatocytegrowth factor (HGF) and an insulin-like growth factor (IFG-1), thehydrogel is at least one selected from the group consisting of fibringlue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose andpectin, the biodegradable support is selected from the group consistingof poly-gamma-glutamic acid (PGA), poly lactic acid (PLA), vicryl mesh,human placental membrane, bovine placental membrane, pig collagen,chitin, chitosan, fibronectin and dextran, and the nondegradable supportis selected from the group consisting of sterilized nonwoven fabrics,polyethylene terephthalate (PET) films, polyethylene (PE) films,polypropylene (PP) films, polyurethane films, net type polyurethanefilms, and polyurethane coated with soft silicon on a single surface.

More specifically, the present invention provides a preparing method ofa sheet for alleviating or improving epidermolysis bullosa, including:(a1) at least 2-subculturing mesenchymal stem cells in a growth medium;(a2) obtaining a mesenchymal stem cells-hydrogel-support by attachingthe cultured mesenchymal stem cells to (i) at least one kind of supportselected from the group consisting of biodegradable supports andnondegradable supports; (ii) at least two kinds of nondegradablesupports; or (iii) a combination of at least one kind of support and atleast one kind of nondegradable support using a hydrogel; and (b)culturing the mesenchymal stem cells-hydrogel-support obtained in step(a2) in a growth medium. Herein, the growth medium is a medium includingfetal bovine serum (FBS) and at least one factor selected from the groupconsisting of a basic fibroblast growth factor (bFGF), an epidermalgrowth factor (EGF), a transforming growth factor beta-1 (TGF-beta1), aplatelet-derived growth factor (PDGF), a vascular endothelial growthfactor (VEGF), a hepatocyte growth factor (HGF) and an insulin-likegrowth factor (IFG-1), the hydrogel is at least one selected from thegroup consisting of fibrin glue, hyaluronic acid, gelatin, collagen,alginic acid, cellulose and pectin, the biodegradable support isselected from the group consisting of poly-gamma-glutamic acid (PGA),poly lactic acid (PLA), vicryl mesh, human placental membrane, bovineplacental membrane, pig collagen, chitin, chitosan, fibronectin anddextran, and the nondegradable support is selected from the groupconsisting of sterilized nonwoven fabrics, polyethylene terephthalate(PET) films, polyethylene (PE) films, polypropylene (PP) films,polyurethane films, net type polyurethane films, and polyurethane coatedwith soft silicon on a single surface.

In one embodiment according to the present invention, the factorincluded in the growth medium may be more specifically, a basicfibroblast growth factor (bFGF), an epidermal growth factor (EGF), or acombination thereof.

In one embodiment according to the present invention, the mesenchymalstem cells are autologous or allogenic cells that are positive for CD29,CD44, CD73, CD90, and CD105 and negative for CD34 and CD45.

In one embodiment according to the present invention, in order toprepare the sheet for alleviating or improving epidermolysis bullosa,the method includes proliferating stem cells of 20,000 or more and morespecifically 20,000 to 400,000 per 1 cm² of the support by mixing thestem cells with the hydrogel to evenly coat 1,000 to 10,000 stem cellsper 1 cm² of the support and culturing the stem cells in a mediumcontaining at least one selected from the group consisting of FBS, bFGFand EGF.

In one embodiment according to the present invention, in step (c), themethod further includes (c) activating the cells by additionallyperforming at least one stimulation selected from the group consistingof physical stimulation, hypoxic stimulation, mitogen stimulation, andinflammatory factor stimulation such as IFN-gamma. In particular, in thecase of tissues damaged by epidermolysis bullosa, since blood vesselsare destroyed or a function is deteriorated, oxygen supply is not smoothand chronic inflammation may exist, stem cells administered to thedamaged tissues are activated by hypoxic stress and inflammatory factorsand the secretion of growth factors and cytokines is rapidly increased.Accordingly, it is possible to prepare the composition or sheetincluding high-active stem cells which secrete growth factors andcytokines at a high concentration by treating hypoxic stress, mitogensor inflammatory factors in the preparing of the support sheet.

In one embodiment according to the present invention, the hydrogel mayuse fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid,cellulose or pectin, but is not limited thereto. When fibrin glue isused as the hydrogel, the concentration of fibrinogen forming fibringlue may be 0.5 to 60 mg/mL, specifically 0.5 to 45 mg/mL, morespecifically 0.5 to 30 mg/mL, much more specifically 0.5 to 20 mg/mL,much more specifically 0.5 to 10 mg/mL, and much more specifically 0.5to 5 mg/mL, and thrombin may be included at a concentration of 1 to 50I.U./mL, specifically 1 to 30 I.U./mL, and more specifically 5 to 20I.U./mL.

In one embodiment according to the present invention, the stemcells-hydrogel sheet may be prepared by only the hydrogel itself, butthe hydrogel has a low strength and may easily be torn bymechanical/physical force, and thus there are disadvantages that thesize of the sheet is limited and careful attention in use is required.When attaching the mesenchymal stem cells-hydrogel to the biodegradablesupport or the nondegradable support, the support enhances the strengthof the mesenchymal stem cells-hydrogel and makes manipulation easier.

In one embodiment of the present invention, the biodegradable supportmay be one kind or a combination of two or more kinds selected from thegroup consisting of poly-gamma-glutamic acid (PGA), poly lactic acid(PLA), vicryl mesh, human placental membrane, bovine placental membrane,pig collagen, chitin, chitosan, fibronectin and dextran, and thenondegradable support may be one kind or a combination of two or morekinds selected from the group consisting of sterilized nonwoven fabrics,polyethylene terephthalate (PET) films, polyurethane films, net typepolyurethane films, polyurethane films coated with soft silicon on asingle surface, polyethylene (PE) films, and polypropylene (PP) films,but is not limited thereto.

In one embodiment of the present invention, a combination of two or morenondegradable supports may be used, but is not limited thereto.

In one embodiment of the present invention, a combination of one or morebiodegradable supports and nondegradable supports may be used as thesupport. For example, a polyurethane film, a net type polyurethane film,a polyurethane film coated with soft silicon on a single surface,PLA/nonwoven fabric, or PGA/PLA/nonwoven fabric may be used, but is notlimited thereto.

In one embodiment of the present invention, in step (b), the method mayfurther include (d) adding and freezing the mesenchymal stemcells-hydrogel-support in a freezing preservative containing 1 to 20 w/v% DMSO and 1 to 80 w/v % human serum albumin, and herein, when thawingafter freezing, a survival rate of the mesenchymal stem cells is 70% ormore.

In one embodiment of the present invention, when the mesenchymal stemcells-hydrogel-support composition according to the present invention isfrozen and thawed, after 1 week, 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,and 12 months, the survival rate of the mesenchymal stem cells may be70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, and 99% or more, more specifically, 90% ormore and 95% or more after 12 months.

Another embodiment of the present invention provides a composition foralleviating or improving epidermolysis bullosa, containing mesenchymalstem cells and a hydrogel; and at least one support selected from thegroup consisting of biodegradable supports and nondegradable supports ora combination of at least one biodegradable support and at least onenondegradable support.

In one embodiment according to the present invention, the hydrogel maybe at least one selected from the group consisting of fibrin glue,hyaluronic acid, gelatin, collagen, alginic acid, cellulose and pectin,but is not limited thereto. When the fibrin glue is used as thehydrogel, the concentration of fibrinogen forming the fibrin glue may be0.5 to 60 mg/mL, specifically 0.5 to 45 mg/mL, more specifically 0.5 to30 mg/mL, much more specifically 0.5 to 20 mg/mL, much more specifically0.5 to 10 mg/mL, and much more specifically 0.5 to 5 mg/mL, and thrombinmay be included at a concentration of 1 to 50 I.U./mL, specifically 1 to30 I.U./mL, and more specifically 5 to 20 I.U./mL.

In one embodiment of the present invention, the biodegradable supportmay be one kind or a combination of two or more kinds selected from thegroup consisting of poly-gamma-glutamic acid (PGA), poly lactic acid(PLA), vicryl mesh, human placental membrane, bovine placental membrane,pig collagen, chitin, chitosan, fibronectin and dextran, and thenondegradable support may be one kind or a combination of two or morekinds selected from the group consisting of sterilized nonwoven fabrics,polyethylene terephthalate (PET) films, polyethylene (PE) films,polypropylene (PP) films, polyurethane films, net type polyurethanefilms, and polyurethane coated with soft silicon on a single surface,but the present invention is not limited thereto.

In one embodiment of the present invention, a combination of two or morenondegradable supports may be used, but is not limited thereto.

In one embodiment of the present invention, a combination of one or morebiodegradable supports and nondegradable supports may be used as thesupport. For example, a polyurethane film, a net type polyurethane film,a polyurethane film coated with soft silicon on a single surface,PLA/nonwoven fabric, or PGA/PLA/nonwoven fabric may be used, but is notlimited thereto.

Yet anther embodiment of the present invention provides a sheet foralleviating or improving epidermolysis bullosa, containing thecomposition as an active ingredient.

In one embodiment of the present invention, the epidermolysis bullosamay be selected from the group consisting of epidermolysis bullosa,acantholysis bullosa, acanthosis bullosa, epidermolysis bullosaacquisita, epidermolysis bullosa hereditaria, epidermolysis bullosaletalis, epidermolysis bullosa tarda, epidermolysis hereditaria tarda,hyperplastic epidermolysis bullosa, keratolysis, localized epidermolysisbullosa, non-scarring epidermolysis bullosa, polydysplasticepidermolysis bullosa, scarring bullosa, simplex epidermolysis bullosa,Weber-Cockayne disease, Dowling-Meara syndrome, Goldscheider's disease,Hallopeau-Siemens disease, Heinrichsbauer syndrome, Herlitz syndrome,and Kobner's disease.

In one embodiment of the present invention, the mesenchymal stem cellsmay be isolated from fat, bone marrow, or umbilical cord blood.

In one embodiment of the present invention, more specifically, themethod may further include:

(a1) subculturing mesenchymal stem cells in a growth medium containingfetal bovine serum (FBS) and a basic fibroblast growth factor (bFGF) oran epidermal growth factor (EGF) as a growth factor two times or more;

(a2) attaching the cultured mesenchymal stem cells to a biodegradable ornondegradable support or a combination thereof using a hydrogel;

(b) culturing the mesenchymal stem cells-hydrogel-biodegradable ornondegradable support in step (a2) in the medium containing FBS and bFGFor EGF as a growth factor for about 5 days to prepare a sheet;

(c) activating cells by additionally performing physical stimulation,hypoxic stimulation, mitogen stimulation, and inflammatory factorstimulation when culturing in step (b);

(d) washing the mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet in step (b) or (c) with a medium from whichthe FBS and the bFGF or EGF as a growth factor are removed;

(e) freezing the mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet in step (b) in a freezing preservativecontaining 10% DMSO and 5% human serum albumin;

(f) removing the freezing preservative by thawing the frozen mesenchymalstem cells-hydrogel-biodegradable or nondegradable support sheet andwashing the sheet with physiological saline; and

(g) attaching the sheet to an applicable site after cutting the sheetaccording to a size of a wounded part requiring dressing in the woundedpart of a patient with epidermolysis bullosa,

and further include (h) covering the sheet with another dressing.

The method will be described in more detail as follows.

In step (a1), mesenchymal stem cells isolated from human adipose tissuesmay be used, and in this case, a method of isolating the mesenchymalstem cells isolated from human adipose tissues and subculturing themesenchymal stem cells two times or more is disclosed in a prior art(Korean Patent Registration No. 1,328,604) and may effectively obtain alarge amount of mesenchymal stem cells within a short time by culturingcells using a growth medium. According to the prior art, the humanadipose-derived mesenchymal stem cells subcultured two times or more areattached to a plastic culture container to keep a form of fibroblastsand are positive for CD10, CD13, CD29, CD44, CD59, CD71, CD90, CD105 andOct4 and negative for CD34, CD45, CD104, CD106 and Stro-1. Also, thehuman adipose-derived mesenchymal stem cells have ability todifferentiate into adipocytes, bone cells, chondrocytes, muscle cells,and neurons in vitro as stem cells. Also, the human adipose-derivedmesenchymal stem cells secrete a variety of growth factors such as VEGF,HGF, TGF-β1, NGF, and IGF and have immunoregulatory ability, and thus,technologies applied to treatment of various diseases have beendeveloped.

In step (a2), the human adipose-derived mesenchymal stem cellssubcultured two times or more in step (a1) are treated with trypsin ordispase to make a single cell, suspended in the hydrogel, and thenevenly sprayed at a concentration of about 5,000/cm² to be attached tothe biodegradable or nondegradable support fabric. Thereafter, the humanadipose-derived mesenchymal stem cells are cultured for 3 to 7 daysusing the growth medium containing 10% FBS and EGF or bFGF. The hydrogelused in the embodiment of the present invention is a fibrin gel, but isnot limited thereto, and may further include collagen, hyaluronic acid,gelatin, alginic acid, cellulose, and pectin.

Further, the biodegradable support used in the embodiment of the presentinvention is vicryl mesh or bovine placental membrane and thenondegradable support is sterilized gauze, a polyurethane film, a nettype polyurethane film, a soft silicone coated polyurethane on a singlesurface, or a PET film, but are not limited thereto, and may usePGA/nonwoven fabric, PGA/PLA/nonwoven fabric, a human placentalmembrane, a collagen membrane, and the like.

In the present invention, the hydrogel primarily performs a function ofattaching the mesenchymal stem cells to the biodegradable ornondegradable support fabric or the polyurethane film, the net typepolyurethane film, the soft silicone coated polyurethane on a singlesurface, the PET film, the PE film, or the PP film and secondarilyprovides a substrate to the mesenchymal stem cells as attachable cellsto attach the cells to the substrate, thereby providing an environmentthat can survive stably. Further, the hydrogel contains a large numberof three-dimensional network pores, and provides an environment in whichFBS, bFGF, or EGF contained in the culture medium passes through thenetwork structure to act on the cells and may proliferate the cells. Thehydrogel has a major influence on the shape and the growth rate of cellsbecause the size, hardness and decomposition rate of the networkstructure depend on the prepared concentration. In the embodiment of thepresent invention, the growth rate of the mesenchymal stem cells isincreased, while the proper hardness of the gel is maintained by usingthe fibrin gel at a final concentration of 0.5 to 10 mg/mL.

In step (b), in the mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet, the mesenchymal stem cells are rapidlyproliferated and increased four times or more for 3 to 7 days and 20,000or more cells per cm² may be included.

In the present invention, the cells proliferated in the hydrogel expressCD29, CD44, CD73, CD90, CD105 which are characteristic of theadipose-derived mesenchymal stem cells and may secrete various growthfactors including VEGF and HGF. Also, the cells have inhibition abilityof TNF-α and IFN-γ, which are representative inflammatory factorssecreted from immune cells. That is, the cells cultured in the hydrogelmaintain characteristics of the mesenchymal stem cells.

Also, in step (b), hypoxic stress, mitogen treatment, and inflammatoryfactor (IFN-γ) treatment may be used in combination. In the case oftissues damaged by epidermolysis bullosa, since blood vessels aredestroyed or a function is deteriorated, oxygen supply is not smooth andchronic inflammation may exist, stem cells administered to the damagedtissues are activated by hypoxic stress and inflammatory factors and thesecretion of growth factors and cytokines is rapidly increased.

As described above, the present invention provides a preparing method ofa sheet including high-active stem cells secreting growth factors andcytokines at a high concentration by treating hypoxic stress, mitogensor inflammatory factors in the preparing of the adipose-derivedmesenchymal stem cells-hydrogel-biodegradable or nondegradable supportsheet.

Further, the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support sheet preparedaccording to the present invention has excellent therapeutic abilitybecause the high-active stem cells may be applied to the wounded part ofa patient with excellent epidermolysis bullosa as it is withoutisolation using protease. Also, since there is no isolation usingprotease, in the culturing, an extracellular matrix such as collagen,laminin, fibronectin, and elastin secreted from the mesenchymal stemcells is wholly present on the hydrogel, thereby further promoting aneffect of alleviating or improving epidermolysis bullosa.

As described above, in step (a2) of the present invention, the stemcells-hydrogel sheet may be prepared only by the hydrogel itself.However, since the hydrogel is low in strength and easily torn bymechanical/physical force, the size of the sheet is limited and carefulattention is required in use. On the other hand, according to thepresent invention, when a stem cells-hydrogel is attached to abiodegradable or nondegradable support, the support enhances thestrength of the stem cells-hydrogel to be easily manipulated. Inaddition, when the thickness of the sheet is 0.1 to 2 mm, the sheet isprevented from being torn when applied to the wounded part, andsufficient cell count is included in the sheet, thereby improving thetherapeutic effect.

In step (d) of the present invention, the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support sheet iswashed 2 to 3 times with physiological saline to remove FBS as ananimal-derived ingredient and may be washed in a serum-free DMEM mediumin order to completely remove the FBS. In the process, the FBS isremoved to minimize side effects that may be caused by theanimal-derived ingredient when the sheet is applied to the human body.

The present invention includes a method of freezing the adipose-derivedmesenchymal stem cells-hydrogel-biodegradable or nondegradable supportsheet. The sheet is added with a freezing preservation configured by asolution of 10% DMSO and 5% human serum albumin to be sufficientlyimmersed in a Cryovac, sealed, and then stored at −80° C. Generally, itis known that when cells isolated by treating protease or the artificialskin (epidermal cells or dermal cells, or the artificial skin composedof both cells) are frozen at −80° C., the cells are damaged and appliedto the wounds to reduce the therapeutic effect (Tissue eng4(4):1403-414, 1988).

However, in the mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet prepared according to the present invention,the hydrogel covers the stem cells to protect the cells from externalimpact and stress, thereby enabling long-term storage without damage onthe cells even at −80° C.

In one embodiment of the present invention, an effective does of one ormore growth factors, cytokines, hormones or extracellular matrixcompounds or proteins useful for enhancing the effect of alleviating orimproving epidermolysis bullosa may be administered with the compositionaccording to the present invention. Specifically, the examples mayinclude GCSF, IL6, IL8, IL10, MCP1, MCP2, tissue factor, bFGF, KGF,VEGF, PLGF, MMP1, MMP9, TIMP1, TIMP2, TGF-β1, HGF, and the like, but arenot limited thereto.

Hereinafter, the present invention will be described in more detailthrough Examples. However, these Examples are just examples of thepresent invention and the scope of the present invention is not limitedthereto.

Example 1: Method of Culturing Human Adipose-Derived Mesenchymal StemCells

Adipose tissue may be usually obtained by liposuction, but is notlimited thereto. Adipose-derived mesenchymal stem cells were isolatedfrom the adipose tissue obtained by liposuction as follows: To removeblood, the adipose tissue was washed, and then added with the samevolume of collagenase solution as the adipose tissue thereto, andreacted at 37° C. in a water bath.

After the centrifugation, the fat layer as a supernatant was removed,and the collagenase solution as the lower layer was carefully isolatedwithout shaking, suspended in a substrate medium, and then centrifugedat 20° C. and 1500 rpm for 5 minutes. At this time, the lower layerserved as the stroma-vascular fraction, and the supernatant was removed.The stroma-vascular fraction was suspended in the substrate medium,inoculated into a culture vessel, and cultured in a 5% CO₂ incubator at37° C. for 24 hours.

After the removal of the culture medium, the cells were washed with aphosphate buffer solution and proliferated using the substrate medium, agrowth medium containing the bFGF at a concentration of 1 ng/mL in thesubstrate medium, or a medium containing EGF at a concentration of 5ng/mL in the substrate medium. The adipose-derived mesenchymal stemcells were grown about 80 to 90% of the culture vessel and then treatedwith trypsin to be isolated and obtained to single cells.

Example 2: Determination of Concentration of Fibrin Glue as Hydrogel

Lyophilized thrombin was added to 1 mL of a calcium chloride solution tobe 400 to 600 I.U. Alternatively, the frozen thrombin was thawed andadjusted to the same concentration, and then used. The lyophilizedthrombin was added with 1 mL of an aprotinin solution or thawed toprepare a undiluted solution and then the undiluted solution was dilutedstepwise to 1:5, 1:10, 1:20, and 1:40. The cells subcultured two timesor more in Example 1 were collected and suspended, mixed with thrombinat a ratio (v/v) of 40 to 50:1, and then mixed with fibrigen dilutedstepwise at 1:1 to form a fibrin gel. When the gel was completelyhardened, the cells were added with a culture medium containing 10% FBSand 1 ng/mL bFGF and cultured in a 5% CO₂ incubator at 37° C. for 5days.

On the 2nd and 5th day of the culture, the cell-fibrin gel mixture wastaken and made into thin slices, stained with 10 μg/mL of acridineorange/ethidium bromide (AO/EtBr), and a form and a survival rate of thecells were measured using a fluorescence microscope. Also, on the 5-thday of the culture, WST-1 was added to measure a cell growth degree.

FIG. 1A is a micrograph showing the forma and the number of stem cellsin a fibrin gel prepared from a undiluted solution or a dilutedfibrinogen solution. In the fibrin gel made from the fibrinogenundiluted solution, most of the cells kept a spherical cell shape untilthe 5th day and were almost not proliferated. On the other hand, as thedilution ratio increased, the cells rapidly formed fibroblast shapes andwere proliferated more. Some dead cells were observed in the fibrin gelprepared from the undiluted fibrinogen, but no dead cells were observedin the fibrin gel prepared with the diluted fibrinogen. That is, thehigh-concentration fibrin gel shows weak cytotoxicity to theadipose-derived mesenchymal stem cells, but the fibrin gel at a dilutionof 1:5 to 1:40 has no cytotoxicity.

FIG. 1B is a graph showing quantitative measurement of the growthactivity of stem cells according to the fibrinogen gel using WST-1, inwhich as the dilution rate increases, the absorbance increases. In otherwords, it can be seen that the stem cells are proliferated best in thefibrin gel prepared by diluting the fibrinogen 20 or 40 times.

Example 3: Preparation of Human Adipose-Derived Mesenchymal StemCells-Hydrogel-Biodegradable or Nondegradable Support Sheet

The mesenchymal stem cells subcultured two times or more in Example 1were collected and suspended in the growth medium. Based on the resultsof Example 2, thrombin was added to the cell suspension to be a final 8to 15 I.U.

The fibrinogen at a concentration of about 3 to 6.5 mg/mL was appliedevenly on vicryl mesh or bovine placental membrane as a biodegradablesupport having a square shape of about 5×5 cm or gauze, polyurethanecoated with soft silicon on a single surface, and a PET film as anondegradable support, as a support. Thereafter, the cell suspensioncontaining thrombin was applied to the support to have about 5,000 cellsper cm², and then the cell-fibrin gel was uniformly formed and attachedto the support. When the fibrin gel was completely hardened, the cellswere added with a growth medium and cultured at 37° C. in a 5% CO₂incubator for 3 to 7 days.

FIG. 2A is a photograph of a sheet which is prepared by mixing humanadipose-derived mesenchymal stem cells with a fibrin hydrogel andattaching and culturing the mesenchymal stem cells-fibrin hydrogel topolyurethane coated with soft silicon on a single surface and a PET filmas nondegradable supports and then observed by a fluorescence microscopeafter AO/EtBr staining. FIG. 2B is a photograph of the sheet frozen at−80° C. which is thawed and then observed by a microscope or afluorescence microscope after AO/EtBr staining. As illustrated in FIG.2B, it was observed that even after thawing after freezing, the cells inthe form of fibroblasts similar to those before freezing were attachedto the support by the hydrogel and proliferated. About 20,000 to 400,000cells per 1 cm² of the sheet were uniformly distributed and 100%survived.

Example 4: Freezing of Human Adipose-Derived Mesenchymal StemCells-Hydrogel-Biodegradable or Nondegradable Support Sheet

The human adipose-derived mesenchymal stem cells-hydrogel-biodegradableor nondegradable support sheet prepared in Example 3 was washed toremove a cell culture medium, put in a Cryovac containing a freezepreservative (a solution containing 10% DMSO and human serum albumin),and then frozen at −80° C. After about 1 month, 3 months, 6 months, 9months, and 12 months, the Cryovac was taken out for each period andimmersed in a constant temperature water bath at 37° C., and then shakento dissolve and remove the freeze preservative. Physiological saline wasadded and then shaken up and down and removed. After the freezepreservative was completely removed, the cells were stained by AO/EtBrto measure a survival rate.

As a result, as illustrated in FIG. 3 , 95% or more of theadipose-derived mesenchymal stem cells survived until 12 months in thesheet prepared according to the present invention.

That is, it is shown that the human adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support sheet preparedaccording to the present invention may be frozen for a long time at −80°C. without the damage on cells as a main active ingredient.

Example 5: Secretion of Growth Factors of Human Adipose-DerivedMesenchymal Stem Cells-Hydrogel-Biodegradable or Nondegradable Support

The human adipose-derived mesenchymal stem cells-hydrogel-biodegradableor nondegradable support sheet in Example 3 or the frozen sheet inExample 4 was thawed, washed with PBS, and then cut with sizes of0.8×0.8 cm, and then two sheets were put in a 24-well plate and addedwith DMEM 1 mL. After culturing in a 5% CO₂ incubator at 37° C. for 72hours, the supernatant was collected and the amounts of VEGF and HGF,which were representative growth factors secreted from the mesenchymalstem cells, were measured by ELISA. As a result, as illustrated in FIG.4A, the sheet secreted the HGF and the VEGF.

As another example, the collected supernatant was analyzed using acytokine array kit associated with angiogenesis. As a result, asillustrated in FIG. 4B, the sheet secreted large amounts of growthfactors including HGF and VEGF promoting angiogenesis and variouscytokines such as serprinE1 (PAI-1), F1 (PDEF), TIMP-1, CXCL8 (IL-8),FGF-2, and DPPIV (CD26). That is, when the mesenchymal stem cellscultured using the hydrogel and the biodegradable or nondegradablesupport prepared according to the present invention were applied to thewounded part, it was found that various growth factors and cytokinespromoting cell proliferation and angiogenesis were continuously secretedto activate peripheral tissue cells and alleviate or improve theepidermolysis bullosa.

Example 6: Immunoregulatory Function of Homogeneous HumanAdipose-Derived Mesenchymal Stem Cells-Hydrogel-Biodegradable orNondegradable Support

The human adipose-derived mesenchymal stem cells-hydrogel-biodegradableor nondegradable support sheet in Example 3 or the frozen sheet inExample 4 was thawed, cut with appropriate sizes, and then one cut sheetwas put in a 24-well plate. In addition, 5×10⁵ peripheral bloodmononuclear cells (PBMCs) obtained from donors having different humanleukocyte antigens (HLA) were added in a 24-well plate. As a positivecontrol, phyto-hemagglutinin as mitogen was added to the PBMCs to causean immune response of the PBMCs. On the 3th day after the start of theresponse, the supernatant was collected and the amount of secreted TNF-αwas measured by an ELISA method.

As yet another Experimental Example, 5×10⁵ PBMCs were put in the 24-wellplate and activated by PHA to cause the immune response, the homogeneoushuman adipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet prepared in Example 3 or 4 was cut withappropriate sizes, and then one cut sheet was added to the plate. On the3th day of the response, the supernatant was collected and the secretionamount of TNF-α was measured.

As a result, as illustrated in FIG. 5 , in the PBMCs activated by PHA,the homogeneous human adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support sheet reduced thesecretion amount of TNF-α by 60% or more. That is, the homogeneous humanadipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support sheet does not cause the immune response. Also,when an excessive immune response occurs, the sheet serves tosignificantly reduce the secretion amount of TNF-α which is secreted inlarge amount by immune cells and serves to increase immunoreactivity.Accordingly, the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support sheet covers thewounded part of epidermolysis bullosa to continuously alleviate theinflammation and promote re-epithelization.

Example 7: ECM Secretion Ability of Human Adipose-Derived MesenchymalStem Cells

Proteins were extracted from the cells obtained in Example 1 andexpression levels of collagen type VII and laminin 5 as causativeproteins of epidermolysis bullosa were measured by a Western blot methodand then compared with expression levels in fibroblasts. Further, theexpression level and the secretion amount were measured by an ELISAmethod from the human adipose-derived mesenchymal stem cells which werea raw material of the sheet prepared in the present invention.

FIG. 6A shows that the in the human adipose-derived mesenchymal stemcells which were a raw material of the sheet prepared in the presentinvention, expression levels of collagen type VII and laminin 5 are highas compared with the fibroblasts. FIG. 6B illustrates results ofquantifying the amounts of collagen type VII expressed and secreted fromthe human adipose-derived mesenchymal stem cells.

As yet another example, the adipose-derived mesenchymal stem cellsprepared in Example 1 were fixed and added with PBS containing acollagen type VII or collagen type I-specific antibody, and then reactedat 37° C. for 1 hour and washed, mounted, and observed by a fluorescencemicroscope.

FIG. 6C is a photograph (×400) showing secretion ability ofextracellular matrix proteins of the adipose-derived mesenchymal stemcells, and as illustrated in FIG. 6C, the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support preparedaccording to the present invention entirely had a positive reaction forcollagen type VII and collagen type I.

Example 8: Evaluation of ECM Secretion and Expression of HumanAdipose-Derived Mesenchymal Stem Cells-Hydrogel-Biodegradable orNondegradable Support

The human adipose-derived mesenchymal stem cells-hydrogel-biodegradableor nondegradable support prepared in Example 3 or 4 was cultured in aDMEM without adding FBS for 3 days and then the culture medium wascollected to measure ECM secretion ability and expression rate by anELISA method.

As a result, as illustrated in FIG. 7A, the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support preparedaccording to the present invention secreted collage types VII and I.

As yet another example, frozen slices of the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support prepared inExample 3 or 4 were made and fixed, added with an extracellular matrixprotein-specific antibody, reacted at 37° C. for 1 hour, washed,mounted, and then observed by a fluorescence microscope.

As a result, as illustrated in FIG. 7B, the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support preparedaccording to the present invention had a positive reaction for collagetype VII, collage type I, collage type V, laminin 5, fibronectin, andlaminin. That is, the cells configuring the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support preparedaccording to the present invention secreted various types ofextracellular matrix proteins in large amount and the secretedextracellular matrix proteins were left in the hydrogel and transplantedinto the body to provide various substrates, thereby alleviating orimproving epidermolysis bullosa.

Example 9: Expression of Collage Type VII of Human Adipose-DerivedMesenchymal Stem Cells and Human Adipose-Derived Mesenchymal StemCells-Hydrogel-Biodegradable or Nondegradable Support

FIG. 8 illustrates results of secretion ability of adipose-derivedmesenchymal stem cells single-layer cultured according to an existingculture method and extracellular matrix proteins of the adipose-derivedmesenchymal stem cells in the sheet, and in the adipose-derivedmesenchymal stem cells-hydrogel-biodegradable or nondegradable supportprepared according to the present invention, collagen type VII wassecreted 6.8 times larger than that of the existing single-layercultured cells.

As the above result, when the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support is attached to thewounded part of a patient with epidermolysis bullosa, theadipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support supplies various extracellular matrix proteins toa skin damaged part, thereby promoting tissue regeneration andre-epithelization.

Example 10: Evaluation of Expression of Collagen Type VII in HumanAdipose-Derived Mesenchymal Stem Cells by Inflammatory Material

The cells obtained in Example 1 were added to a 12-well plate at5,500/cm², and after 24 hours, TGF-β2 and TNF-α were added and furthercultured for 48 hours. After the cells were collected, RNA was extractedand the expression level of collagen type VII, which was a causativegene of epidermolysis bullosa, was measured by a RT-PCT method and theexpression changes were compared with each other.

As a result, as illustrated in FIG. 9 , when TGF-β2 and TNF-α weretreated to the adipose-derived mesenchymal stem cells as a mainingredient of the sheet, the expression of collagen type VII increasedabout 8 times. As the above result, when the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support preparedaccording to the present invention is attached to the wounded part of apatient with epidermolysis bullosa, the expression level of collagentype VII of the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support is increased byinflammatory materials existing in a chronic skin damaged part, therebypromoting tissue regeneration and re-epithelization.

Example 11: Restoring the Morphology, Adherence Ability, Mobility ofCollagen Type VII Deficient Fibroblasts by Human Adipose-DerivedMesenchymal Stem Cells-Hydrogel-Biodegradable or Nondegradable Support

In order to evaluate whether the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support in Example 4 canrestore the function of collagen type VII deficient cells, fibroblastswere treated with siRNA to induce deficiency of collagen type VII.

As illustrated in FIG. 10 , the collagen type VII deficient fibroblastskept in a round shape without being attached to the bottom of theculture vessel after 24 hours being inoculated into the culture vessel.However, as a result of treating 72 hours of conditioned media (CM) ofthe adipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support, the collagen type VII deficient cells adhered tothe bottom of the culture vessel and the collagen type VII deficientcells restored the fusiform shape similar to normal cells.

As another example, fibroblasts were attached to the bottom of theculture vessel and cultured, and then treated with siRNA to inducecollagen type VII deficiency. As a result that scratch (wound) model wasmade by scraping the center of the cultured cells and mobility of thescratch model to scratch area was evaluated, the mobility of collagentype VII deficient fibroblasts was decreased compared to normalfibroblasts. Here, when treating adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support CM, migration ofcollagen type VII deficient cells was promoted as shown in FIG. 11 .

As the above result, it can be seen that the adipose-derived mesenchymalstem cells-hydrogel-biodegradable or nondegradable support can restorethe function of collagen type VII deficient cells and effectivelyrestore the skin tissue.

Example 12: Treatment Example of Epidermolysis Bullosa by HumanAdipose-Derived Mesenchymal Stem Cells-Hydrogel-Biodegradable orNondegradable Support

In order to evaluate a therapeutic effect of the adipose-derivedmesenchymal stem cells-hydrogel-biodegradable or nondegradable supportprepared in Example 4, clinical trials were conducted in a patient withepidermolysis bullosa.

This patient was a 24-year-old female patient and diagnosed asepidermolysis bullosa, but since there was not separate therapeuticmethod, there was no improvement in the disease due to continuousrecurrence and inflammation. The adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support prepared inExample 4 was attached to the wounded part of the patient andphotographed and observed at an interval of 1 week, and then a woundtreatment effect was evaluated.

FIG. 12 is a graph showing a wound area after attaching theadipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support, and as the time elapsed after attaching theadipose-derived mesenchymal stem cells-hydrogel-biodegradable ornondegradable support prepared according to the present invention, thewound area was reduced, and after 4 weeks of the attachment, the woundarea was reduced by 64.4% as compared with before attachment.

That is, it can be seen that the adipose-derived mesenchymal stemcells-hydrogel-biodegradable or nondegradable support prepared accordingto the present invention secreted various types of extracellular matrixproteins in large amount, and the secreted extracellular matrix proteinswere left in the hydrogel and transplanted into the body to providevarious substrates, thereby alleviating or improving epidermolysisbullosa.

INDUSTRIAL AVAILABILITY

The composition or sheet for alleviating or improving epidermolysisbullosa, containing the mesenchymal stem cells-hydrogel-biodegradablesupport or nondegradable support according to the present inventionincreases the expression of specifically, collagen type VII andlaminin-5 to express a clinically significant effect when being appliedto the wounded part of epidermolysis bullosa. Therefore, the compositionor sheet may be usefully used for alleviating or improving symptoms of apatient with epidermolysis bullosa.

1-20. (canceled)
 21. A method for alleviating or improving epidermolysisbullosa in a subject in need thereof, comprising attaching anadipose-derived mesenchymal stem cells-hydrogel support sheet to anaffected area of epidermolysis bullosa of the subject in need thereof,wherein the adipose-derived mesenchymal stem cells-hydrogel supportsheet comprises: adipose-derived mesenchymal stem cells; a hydrogel; andat least one support selected from the group consisting of abiodegradable support and a nondegradable support, at least twonondegradable supports, and a combination of at least one biodegradablesupport and at least one nondegradable support; wherein theadipose-derived mesenchymal stem cells and the hydrogel are attached tothe at least one support; wherein the hydrogel is selected from thegroup consisting of fibrin glue, hyaluronic acid, gelatin, collagen,alginic acid, cellulose, and pectin; wherein the biodegradable supportis selected from the group consisting of poly-gamma-glutamic acid (PGA),poly lactic acid (PLA), vicryl mesh, human placental membrane, bovineplacental membrane, pig collagen, chitin, chitosan, fibronectin, anddextran, and wherein the nondegradable support is selected from thegroup consisting of sterilized nonwoven fabrics, polyethyleneterephthalate (PET) films, polyethylene (PE) films, polypropylene (PP)films, polyurethane films, net type polyurethane films, and polyurethanecoated with soft silicon on a single surface.
 22. The method of claim21, wherein the adipose-derived mesenchymal stem cells-hydrogel supportsheet is not pre-treated with TGF-β2 and TNF-α.
 23. The method of claim21, wherein the adipose-derived mesenchymal stem cells-hydrogel supportsheet comprises an extracellular matrix containing collagen type VII andlaminin-5 secreted from the adipose-derived mesenchymal stem cells. 24.The method of claim 21, wherein the hydrogel is fibrin glue comprisingfibrinogen at a concentration of 0.5 to 45 mg/mL.
 25. The method ofclaim 24, wherein the fibrin glue comprises fibrinogen at aconcentration of 0.5 to 10 mg/mL.
 26. The method of claim 21, whereinthe adipose-derived mesenchymal stem cells-hydrogel support sheetcomprises 20,000 to 400,000 of the adipose-derived mesenchymal stemcells per 1 cm² of the support.
 27. The method of claim 21, wherein theepidermolysis bullosa is selected from the group consisting ofepidermolysis bullosa, acantholysis bullosa, acanthosis bullosa,epidermolysis bullosa acquisita, epidermolysis bullosa hereditaria,epidermolysis bullosa letalis, epidermolysis bullosa tarda,epidermolysis hereditaria tarda, hyperplastic epidermolysis bullosa,keratolysis, localized epidermolysis bullosa, non-scarring epidermolysisbullosa, polydysplastic epidermolysis bullosa, scarring bullosa, simplexepidermolysis bullosa, Weber-Cockayne disease, Dowling-Meara syndrome,Goldscheider's disease, Hallopeau-Siemens disease, Heinrichsbauersyndrome, Herlitz syndrome, and Kobner's disease.
 28. The method ofclaim 21, wherein the adipose-derived mesenchymal stem cells on theadipose-derived mesenchymal stem cells-hydrogel support sheet have asurvival rate of 90% or more when thawing after freezing for 12 monthsor more.